Connecting Circadian Clock Dynamics to Metabolism and Fitness

Project Summary The Rust Lab seeks to understand the biophysical basis of circadian rhythms and how they are connected to metabolism to support health and fitness. To do this, we are leveraging the power of a bacterial model system where the circadian clock functions cell autonomously and the core oscillator can be reconstituted in a test tube. We use a combination of live cell microscopy, protein biochemistry, and deep sequencing approaches to interrogate this system. The ultimate goal of this research program is to understand quantitatively the relationship between the circadian clock and the external environment that is required for fitness and to be able to recreate an oscillatory system that has these properties using purified components. During the award period, we plan to build on our previous discoveries linking clock control of glycogen metabolism, protein solubility, and DNA replication to understand how an appropriately timed metabolic switch allows cells to tolerate cycling environments. To do this, we are creating new technologies that will enable new assays. We will extend existing protein reconstitution systems using computer-controlled liquid handling to create oscillators that mimic protein synthesis and degradation characteristic of growing cells. We are developing live cell fluorescence reporters of glycogen metabolism. Finally, we will unleash the power of deep scanning mutagenesis and high-throughput sequencing on circadian clocks by creating saturating point mutant libraries of clock genes. We will determine the oscillator phenotype of each mutation as well as its fitness in cycling environments. This will provide an unprecedented view of the mutational plasticity of the circadian rhythm, the molecular determinants of the rhythm, and the selective pressures on the clock.